The present invention relates to an improved method for detecting the presence of Pseudomonas aeruginosa (P. aeruginosa) in a fluid sample. More specifically, it relates to a method of using specific primers in a polymerase chain reaction (PCR) process to clone a segment of the P. aeruginosa exotoxin A (ETA) gene having at least 400 base pairs.
P. aeruginosa is an opportunistic pathogen capable of producing two different ADP-ribosyltransferase toxins: ETA and exoenzyme S. Exoenzyme S can cause significant tissue damage in lung, burn and wound infections in both humans and animals. Exotoxin A is highly toxic and is produced by a majority of P. aeruginosa strains. The toxin is known to inhibit eucaryotic protein biosynthesis at the level of peptide chain elongation, which is similar to diphtheria toxin. It has been observed that as few as 10 to 100 cells of P. aeruginosa can lead to intestinal colonization in patients who are immunosuppressed. A few illustrative causes of immunosuppression include transplant patients, AIDS patients, chemotherapy recipients, and the like.
Since P. aeruginosa is medically important, various methods have been developed to identify P. aeruginosa species. Such methods include, but are not limited to, using monoclonal antibodies, immunofluorescent antibodies, conventional microbiological methods and DNA or RNA sequencing replication. Each of these methods has, to a limited extent, a disadvantage. The immunofluorescent-antibody test is unreliable because it produces a dull olive green or yellow color which is hard to distinguish from autofluorescense. Conventional microbiological methods, although accurate, are very time intensive since active organisms must be cultured and isolated. This usually requires several days under ideal conditions. Using DNA or RNA sequencing techniques require a measurable amount of DNA or RNA to work. Plus, the probe chosen must be specific enough so as to exclude other organisms that may have a similar nucleic acid sequence. To overcome these disadvantages, DNA/RNA primers and probes have had to have a substantial number of nucleotides to be specific to a targeted gene fragment within the organism.
To overcome the disadvantages associated with the low amount of DNA/RNA present in a test sample PCR can be used, under hybridizing conditions, to multiply the targeted gene core fragment. PCR synthetically increases the nucleotide material present without necessarily having a large number of organisms initially to derive starting genetic material.
Briefly, PCR involves treating a DNA sample, under hybridizing conditions, with one oligonucleotide primer for each strand of each different specific sequence suspected of being present in the sample. Using the primers and agents for polymerization, an extension product of each primer is synthesized which is complementary to each nucleic acid strand. The primer or primers are selected to be substantially complementary to each targeted strand of each specific sequence such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product. The hybridized core fragment is then denatured, generally using heat, to separate the primer extension products. These products are again subjected to complementary nucleotide primers such that a primer extension product is synthesized using each of the single strands produced as a template, resulting in amplification of the specific nucleic acid sequence or sequences if present. This process is cycled sufficiently to increase the amount of targeted DNA so that detection is possible.
A disadvantage of using PCR is that the oligonucleotide primers must be sufficiently long to specifically hybridize a targeted segment of the nucleotide fragment which itself must be unique to the targeted organism. If the oligonucleotide primers are too long, there is a possibility that the primers will autopolymerize reducing the efficacy of the selected primers. If the oligonucleotide primers are not long enough then they may not be specific to the targeted fragment or organism leading to no differentiation between a targeted organism and a non-targeted organism. Accordingly, there is a need for a primer for detecting P. aeruginosa which will reduce or eliminate autopolymerization and have a high sensitivity for the target organism, P. aeruginosa.